Compositions and methods for modulation of smn2 splicing

ABSTRACT

Disclosed herein are compounds, compositions and methods for modulating splicing of SMN2 mRNA in a cell, tissue or animal. Also provided are uses of disclosed compounds and compositions in the manufacture of a medicament for treatment of diseases and disorders, including spinal muscular atrophy.

INCORPORATION OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledCORE0058USC3SEQ_ST25.txt, created on Sep. 14, 2016 which is 24 Kb insize. The information in the electronic format of the sequence listingis incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Newly synthesized eukaryotic mRNA molecules, also known as primarytranscripts or pre-mRNA, made in the nucleus, are processed before orduring transport to the cytoplasm for translation. Processing of thepre-mRNAs includes addition of a 5′ methylated cap and an approximately200-250 base poly(A) tail to the 3′ end of the transcript.

The next step in mRNA processing is splicing of the pre-mRNA, whichoccurs in the maturation of 90-95% of mammalian mRNAs. Introns (orintervening sequences) are regions of a primary transcript (or the DNAencoding it) that are not included in the coding sequence of the maturemRNA. Exons are regions of a primary transcript that remain in themature mRNA when it reaches the cytoplasm. The exons are splicedtogether to form the mature mRNA sequence. Splice junctions are alsoreferred to as splice sites with the 5′ side of the junction oftencalled the “5′ splice site,” or “splice donor site” and the 3′ side the“3′ splice site” or “splice acceptor site.” In splicing, the 3′ end ofan upstream exon is joined to the 5′ end of the downstream exon. Thusthe unspliced RNA (or pre-mRNA) has an exon/intron junction at the 5′end of an intron and an intron/exon junction at the 3′ end of an intron.After the intron is removed, the exons are contiguous at what issometimes referred to as the exon/exon junction or boundary in themature mRNA. Cryptic splice sites are those which are less often usedbut may be used when the usual splice site is blocked or unavailable.Alternative splicing, defined as the splicing together of differentcombinations of exons, often results in multiple mRNA transcripts from asingle gene.

Up to 50% of human genetic diseases resulting from a point mutation arecaused by aberrant splicing. Such point mutations can either disrupt acurrent splice site or create a new splice site, resulting in mRNAtranscripts comprised of a different combination of exons or withdeletions in exons. Point mutations also can result in activation of acryptic splice site or disrupt regulatory cis elements (i.e. splicingenhancers or silencers) (Cartegni et al., Nat. Rev. Genet., 2002, 3,285-298; Drawczak et al., Hum. Genet., 1992, 90, 41-54).

Antisense oligonucleotides have been used to target mutations that leadto aberrant splicing in several genetic diseases in order to redirectsplicing to give a desired splice product (Kole, Acta BiochimicaPolonica, 1997, 44, 231-238). Such diseases include β-thalassemia(Dominski and Kole, Proc. Natl. Acad. Sci. USA, 1993, 90, 8673-8677;Sierakowska et al., Nucleosides & Nucleotides, 1997, 16, 1173-1182;Sierakowska et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 12840-44;Lacerra et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 9591-9596);dystrophin Kobe (Takeshima et al., J. Clin. Invest., 1995, 95, 515-520);Duchenne muscular dystrophy (Dunckley et al. Nucleosides & Nucleotides,1997, 16, 1665-1668; Dunckley et al. Human Mol. Genetics, 1998, 5,1083-90); osteogenesis imperfecta (Wang and Marini, J. Clin Invest.,1996, 97, 448-454); and cystic fibrosis (Friedman et al., J. Biol.Chem., 1999, 274, 36193-36199).

Antisense compounds have also been used to alter the ratio of the longand short forms of Bcl-x pre-mRNA (U.S. Pat. No. 6,172,216; U.S. Pat.No. 6,214,986; Taylor et al., Nat. Biotechnol. 1999, 17, 1097-1100) orto force skipping of specific exons containing premature terminationcodons (Wilton et al., Neuromuscul. Disord., 1999, 9, 330-338). U.S.Pat. No. 5,627,274 and WO 94/26887 disclose compositions and methods forcombating aberrant splicing in a pre-mRNA molecule containing a mutationusing antisense oligonucleotides which do not activate RNAse H.

Proximal spinal muscular atrophy (SMA) is a genetic, neurodegenerativedisorder characterized by the loss of spinal motor neurons. SMA is anautosomal recessive disease of early onset and is currently the leadingcause of death among infants. The severity of SMA varies among patientsand has thus been classified into three types. Type I SMA is the mostsevere form with onset at birth or within 6 months and typically resultsin death within 2 years. Children with type I SMA are unable to sit orwalk. Type II SMA is the intermediate form and patients are able to sit,but cannot stand or walk. Patients with type III SMA, a chronic form ofthe disease, typically develop SMA after 18 months of age (Lefebvre etal., Hum. Mol. Genet., 1998, 7, 1531-1536).

SMA is caused by the loss of both copies of survival of motor neuron 1(SMN1), a protein that is part of a multi-protein complex thought to beinvolved in snRNP biogenesis and recycling. A nearly identical gene,SMN2, exists in a duplicated region on chromosome 5q13. Although SMN1and SMN2 have the potential to code for the same protein, SMN2 containsa translationally silent mutation at position +6 of exon 7, whichresults in inefficient inclusion of exon 7 in SMN2 transcripts. Thus,the predominant form of SMN2 is a truncated version, lacking exon 7,which is unstable and inactive (Cartegni and Krainer, Nat. Genet., 2002,30, 377-384).

Chimeric peptide nucleic acid molecules designed to modulate splicing ofSMN2 have been described (WO 02/38738; Cartegni and Krainer, Nat.Struct. Biol., 2003, 10, 120-125).

Antisense technology is an effective means for modulating the expressionof one or more specific gene products, including alternative spliceproducts, and is uniquely useful in a number of therapeutic, diagnostic,and research applications. The principle behind antisense technology isthat an antisense compound, which hybridizes to a target nucleic acid,modulates gene expression activities such as transcription, splicing ortranslation through one of a number of antisense mechanisms. Thesequence specificity of antisense compounds makes them extremelyattractive as tools for target validation and gene functionalization, aswell as therapeutics to selectively modulate the expression of genesinvolved in disease.

Disclosed herein are antisense compounds useful for modulating geneexpression and associated pathways via antisense mechanisms, which mayinclude antisense mechanisms based on target occupancy. Provided hereinare antisense compounds targeting SMN2 for use in modulation of SMN2splicing. One having skill in the art, once armed with this disclosurewill be able, without undue experimentation, to identify, prepare andexploit antisense compounds for these uses.

SUMMARY OF THE INVENTION

The present invention is directed to antisense compounds targeted to andhybridizable with a nucleic acid molecule encoding SMN2. Provided areantisense compounds targeted to intron, 6, exon 7 or intron 7 of SMN2which modulate splicing of SMN2 pre-mRNAs. In one embodiment, modulationof splicing results in an increase in exon 7 inclusion. In anotherembodiment, modulation of splicing results in a decrease in exon 7inclusion. Contemplated and provided herein are antisense compounds 12to 20 nucleotides in length targeted to intron 6, exon 7 or intron 7 ofSMN2, wherein the compounds comprise 2′-O-methoxyethyl sugarmodifications.

In one aspect of the invention, the antisense compounds are targeted tocis splicing regulatory elements. Regulatory elements include exonicsplicing enhancers, exonic splicing silencers, intronic splicingenhancers and intronic splicing silencers. Exonic and intronic splicingsilencers are preferred targets.

In one embodiment, the antisense compounds comprise at least an8-nucleobase portion of one of the exemplary compounds provided herein.

Also provided are methods for modulating splicing of SMN2 mRNA in acell, tissue or organ using one or more of the compounds of theinvention. In one embodiment, modulation of splicing is exon inclusion.In another embodiment, modulation of splicing is exon skipping. In oneaspect, the compound is targeted to an intronic splicing silencerelement. In another aspect, the compound is targeted to an exonicsplicing silencer element.

Further provided are antisense compounds 10 to 50, 12 to 30 or 12 to 20nucleotides in length targeted to intron 6, exon 7 or intron 7 of SMN2comprising 2′-O-methoxyethyl sugar modifications for use in therapy.Also provided are pharmaceutical compositions comprising one or more ofthe compounds of the invention. Use of an antisense oligonucleotideprovided herein for the preparation of a medicament for modulatingsplicing of an SMN2 pre-mRNA is also provided. In one aspect, modulationof splicing results in an increase in exon 7 inclusion. Use of anantisense oligonucleotide provided herein for the preparation of amedicament for the treatment of spinal muscular atrophy is furtherprovided.

DETAILED DESCRIPTION OF THE INVENTION

Antisense technology is an effective means for modulating the expressionof one or more specific gene products and is uniquely useful in a numberof therapeutic, diagnostic, and research applications. Provided hereinare antisense compounds useful for modulating gene expression viaantisense mechanisms of action, including antisense mechanisms based ontarget occupancy. In one aspect, the antisense compounds provided hereinmodulate splicing of a target gene. Such modulation includes promotingor inhibiting exon inclusion. Further provided herein are antisensecompounds targeted to cis splicing regulatory elements present inpre-mRNA molecules, including exonic splicing enhancers, exonic splicingsilencers, intronic splicing enhancers and intronic splicing silencers.Disruption of cis splicing regulatory elements is thought to altersplice site selection, which may lead to an alteration in thecomposition of splice products.

Processing of eukaryotic pre-mRNAs is a complex process that requires amultitude of signals and protein factors to achieve appropriate mRNAsplicing. Exon definition by the spliceosome requires more than thecanonical splicing signals which define intron-exon boundaries. One suchadditional signal is provided by cis-acting regulatory enhancer andsilencer sequences. Exonic splicing enhancers (ESE), exonic splicingsilencers (ESS), intronic splicing enhancers (ISE) and intron splicingsilencers (ISS) have been identified which either repress or enhanceusage of splice donor sites or splice acceptor sites, depending on theirsite and mode of action (Yeo et al. 2004, Proc. Natl. Acad. Sci. U.S.A.101(44):15700-15705). Binding of specific proteins (trans factors) tothese regulatory sequences directs the splicing process, eitherpromoting or inhibiting usage of particular splice sites and thusmodulating the ratio of splicing products (Scamborova et al. 2004, Mol.Cell. Biol. 24(5):1855-1869; Hovhannisyan and Carstens, 2005, Mol. Cell.Biol. 25(1):250-263; Minovitsky et al. 2005, Nucleic Acids Res.33(2):714-724). Little is known about the trans factors that interactwith intronic splicing elements; however, several studies have providedinformation on exonic splicing elements. For example, ESEs are known tobe involved in both alternative and constitutive splicing by acting asbinding sites for members of the SR protein family. SR proteins bind tosplicing elements via their RNA-binding domain and promote splicing byrecruiting spliceosomal components with protein-protein interactionsmediated by their RS domain, which is comprised of several Arg-Serdipeptides (Cartegni and Krainer, 2003, Nat. Struct. Biol.10(2):120-125; Wang et al. 2005, Nucleic Acids Res. 33(16):5053-5062).ESEs have been found to be enriched in regions of exons that are closeto splice sites, particularly 80 to 120 bases from the ends of spliceacceptor sites (Wu et al. 2005, Genomics 86:329-336). Consensussequences have been determined for four members of the SR proteinfamily, SF2/ASF, SC35, SRp40 and SRp55 (Cartegni et al. 2003, NucleicAcids Res. 31(13):3568-3571).

Although the trans factors that bind intronic splicing regulatoryelements have not been extensively studied, SR proteins andheterogeneous ribonucleoproteins (hnRNPs) have both been suggested tointeract with these elements (Yeo et al. 2004, Proc. Natl. Acad. Sci.U.S.A. 101(44):15700-15705). Two intronic splicing enhancer elements(ISEs) have been identified in SMN2, one in intron 6 and the other inintron 7 (Miyajima et al. 2002, J. Biol. Chem. 22:23271-23277). Gelshift assays using the ISE in intron 7 showed formation of RNA-proteincomplexes, which suggests these trans proteins may be important forregulation of splicing (Miyaso et al. 2003, J. Biol. Chem.278(18):15825-15831).

The role of SMN2 in diseases such as spinal muscular atrophy (SMA) makesit an important therapeutic target. SMA is a genetic disordercharacterized by degeneration of spinal motor neurons. SMA is caused bythe loss of both functional copies of SMN1. However, SMN2 has thepotential to code for the same protein as SMN1 and thus overcome thegenetic defect of SMA patients. SMN2 contains a translationally silentmutation (C→T) at position +6 of exon 7 (nucleotide 66 of SEQ ID NO: 1),which results in inefficient inclusion of exon 7 in SMN2 transcripts.Therefore, the predominant form of SMN2, one which lacks exon 7, isunstable and inactive. Thus, therapeutic compounds capable of modulatingSMN2 splicing such that the percentage of SMN2 transcripts containingexon 7 is increased, would be useful for the treatment of SMA.

Overview

Disclosed herein are oligomeric compounds, including antisenseoligonucleotides and other antisense compounds for use in modulating theexpression of nucleic acid molecules encoding SMN2. This is accomplishedby providing oligomeric compounds which hybridize with one or moretarget nucleic acid molecules encoding SMN2. As used herein, the terms“target nucleic acid” and “nucleic acid molecule encoding SMN2” havebeen used for convenience to encompass DNA encoding SMN2, RNA (includingpre-mRNA and mRNA or portions thereof) transcribed from such DNA, andalso cDNA derived from such RNA.

Provided herein are antisense compounds for use in modulation of SMN2pre-mRNA splicing. In one embodiment, the disclosed antisense compoundsare targeted to exon 7 of SMN2 such that SMN mRNA splicing is modulated.In another embodiment, the antisense compounds are targeted to intron 6of SMN2. In another embodiment, the antisense compounds are targeted tointron 7 of SMN2. Modulation of splicing may result in exon 7 inclusionor exon 7 skipping.

Also provided are antisense compounds targeted to cis regulatoryelements. In one embodiment, the regulatory element is in an exon. Inanother embodiment, the regulatory element is an in intron.

Modulation of Splicing

As used herein, modulation of splicing refers to altering the processingof a pre-mRNA transcript such that the spliced mRNA molecule containseither a different combination of exons as a result of exon skipping orexon inclusion, a deletion in one or more exons, or additional sequencenot normally found in the spliced mRNA (e.g., intron sequence). In thecontext of the present invention, modulation of splicing refers toaltering splicing of SMN2 pre-mRNA to achieve exon skipping or exoninclusion. In one embodiment, exon skipping results in an SMN2 mRNAtranscript lacking exon 7 and exon inclusion results in an SMN2 mRNAtranscript containing exon 7.

As used herein, alternative splicing is defined as the splicing togetherof different combinations of exons, which may result in multiple mRNAtranscripts from a single gene. In the context of the present invention,an SMN2 mRNA transcript containing exon 7 and an SMN2 mRNA transcriptlacking exon 7 are two products of alternative splicing.

Compounds

The term “oligomeric compound” refers to a polymeric structure capableof hybridizing to a region of a nucleic acid molecule. This termincludes oligonucleotides, oligonucleosides, oligonucleotide analogs,oligonucleotide mimetics and chimeric combinations of these. An“antisense compound” or “antisense oligomeric compound” refers to anoligomeric compound that is at least partially complementary to theregion of a nucleic acid molecule to which it hybridizes and whichmodulates its expression. Consequently, while all antisense compoundscan be said to be oligomeric compounds, not all oligomeric compounds areantisense compounds. An “antisense oligonucleotide” is an antisensecompound that is a nucleic acid-based oligomer. An antisenseoligonucleotide can be chemically modified. Nonlimiting examples ofoligomeric compounds include primers, probes, antisense compounds,antisense oligonucleotides, external guide sequence (EGS)oligonucleotides, alternate splicers, and siRNAs. As such, thesecompounds can be introduced in the form of single-stranded,double-stranded, circular, branched or hairpins and can containstructural elements such as internal or terminal bulges or loops.Oligomeric double-stranded compounds can be two strands hybridized toform double-stranded compounds or a single strand with sufficient selfcomplementarity to allow for hybridization and formation of a fully orpartially double-stranded compound.

The oligomeric compounds in accordance with this invention may comprisea complementary oligomeric compound from about 10 to about 50nucleobases (i.e. from about 10 to about 50 linked nucleosides). Onehaving ordinary skill in the art will appreciate that this embodiesantisense compounds of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases.

In one embodiment, the antisense compounds of the invention are 12 to 30nucleobases. One having ordinary skill in the art will appreciate thatthis embodies antisense compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobases.

In one embodiment, the antisense compounds of the invention are 12 to 20nucleobases. One having ordinary skill in the art will appreciate thatthis embodies antisense compounds of 12, 13, 14, 15, 16, 17, 18, 19 or20 nucleobases.

In one embodiment, the antisense compounds of the invention haveantisense portions of 20 nucleobases.

In one embodiment, the antisense compounds of the invention haveantisense portions of 18 nucleobases.

In one embodiment, the antisense compounds of the invention haveantisense portions of 15 nucleobases.

In one embodiment, the antisense compounds of the invention haveantisense portions of 12 nucleobases.

Antisense compounds 10-50 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

Compounds of the invention include oligonucleotide sequences thatcomprise at least the 8 consecutive nucleobases from the 5′-terminus ofone of the illustrative antisense compounds (the remaining nucleobasesbeing a consecutive stretch of nucleobases continuing upstream of the5′-terminus of the antisense compound until the oligonucleotide containsabout 10 to about 50 nucleobases). Other compounds are represented byoligonucleotide sequences that comprise at least the 8 consecutivenucleobases from the 3′-terminus of one of the illustrative antisensecompounds (the remaining nucleobases being a consecutive stretch ofnucleobases continuing downstream of the 3′-terminus of the antisensecompound and continuing until the oligonucleotide contains about 10 toabout 50 nucleobases). It is also understood that compounds may berepresented by oligonucleotide sequences that comprise at least 8consecutive nucleobases from an internal portion of the sequence of anillustrative compound, and may extend in either or both directions untilthe oligonucleotide contains about 10 to about 50 nucleobases. Thecompounds described herein are specifically hybridizable to the targetnucleic acid.

One having skill in the art armed with the antisense compoundsillustrated herein will be able, without undue experimentation, toidentify further antisense compounds.

Hybridization

As used herein, “hybridization” means the pairing of complementarystrands of antisense compounds to their target sequence. While notlimited to a particular mechanism, the most common mechanism of pairinginvolves hydrogen bonding, which may be Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary nucleoside ornucleotide bases (nucleobases). For example, the natural base adenine iscomplementary to the natural nucleobases thymidine and uracil which pairthrough the formation of hydrogen bonds. The natural base guanine iscomplementary to the natural bases cytosine and 5-methyl cytosine.Hybridization can occur under varying circumstances.

An antisense compound is specifically hybridizable when there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

As used herein, “stringent hybridization conditions” or “stringentconditions” refers to conditions under which an antisense compound willhybridize to its target sequence, but to a minimal number of othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances, and “stringent conditions” underwhich antisense compounds hybridize to a target sequence are determinedby the nature and composition of the antisense compounds and the assaysin which they are being investigated.

Complementarity

“Complementarity,” as used herein, refers to the capacity for precisepairing between two nucleobases on either two oligomeric compoundstrands or an antisense compound with its target nucleic acid. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to be acomplementary position.

“Complementarity” can also be viewed in the context of an antisensecompound and its target, rather than in a base by base manner. Theantisense compound and the further DNA or RNA are complementary to eachother when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the antisense compound and a targetnucleic acid. One skilled in the art recognizes that the inclusion ofmismatches is possible without eliminating the activity of the antisensecompound. The invention is therefore directed to those antisensecompounds that may contain up to about 20% nucleotides that disrupt basepairing of the antisense compound to the target. Preferably thecompounds contain no more than about 15%, more preferably not more thanabout 10%, most preferably not more than 5% or no mismatches. Theremaining nucleotides do not disrupt hybridization (e.g., universalbases).

It is understood in the art that incorporation of nucleotide affinitymodifications may allow for a greater number of mismatches compared toan unmodified compound. Similarly, certain oligonucleotide sequences maybe more tolerant to mismatches than other oligonucleotide sequences. Oneof the skill in the art is capable of determining an appropriate numberof mismatches between oligonucleotides, or between an oligonucleotideand a target nucleic acid, such as by determining melting temperature.

Identity

Antisense compounds, or a portion thereof, may have a defined percentidentity to a SEQ ID NO, or a compound having a specific Isis number. Asused herein, a sequence is identical to the sequence disclosed herein ifit has the same nucleobase pairing ability. For example, a RNA whichcontains uracil in place of thymidine in the disclosed sequences of theinstant invention would be considered identical as they both pair withadenine. This identity may be over the entire length of the oligomericcompound, or in a portion of the antisense compound (e.g., nucleobases1-20 of a 27-mer may be compared to a 20-mer to determine percentidentity of the oligomeric compound to the SEQ ID NO.) It is understoodby those skilled in the art that an antisense compound need not have anidentical sequence to those described herein to function similarly tothe antisense compound described herein. Shortened versions of antisensecompound taught herein, or non-identical versions of the antisensecompound taught herein fall within the scope of the invention.Non-identical versions are those wherein each base does not have thesame pairing activity as the antisense compounds disclosed herein. Basesdo not have the same pairing activity by being shorter or having atleast one abasic site. Alternatively, a non-identical version caninclude at least one base replaced with a different base with differentpairing activity (e.g., G can be replaced by C, A, or T). Percentidentity is calculated according to the number of bases that haveidentical base pairing corresponding to the SEQ ID NO or antisensecompound to which it is being compared. The non-identical bases may beadjacent to each other, dispersed through out the oligonucleotide, orboth.

For example, a 16-mer having the same sequence as nucleobases 2-17 of a20-mer is 80% identical to the 20-mer. Alternatively, a 20-mercontaining four nucleobases not identical to the 20-mer is also 80%identical to the 20-mer. A 14-mer having the same sequence asnucleobases 1-14 of an 18-mer is 78% identical to the 18-mer. Suchcalculations are well within the ability of those skilled in the art.

The percent identity is based on the percent of nucleobases in theoriginal sequence present in a portion of the modified sequence.Therefore, a 30 nucleobase antisense compound comprising the fullsequence of the complement of a 20 nucleobase active target segmentwould have a portion of 100% identity with the complement of the 20nucleobase active target segment, while further comprising an additional10 nucleobase portion. In the context of the invention, the complementof an active target segment may constitute a single portion. In apreferred embodiment, the oligonucleotides of the instant invention areat least about 80%, more preferably at least about 85%, even morepreferably at least about 90%, most preferably at least 95% identical toat least a portion of the complement of the active target segmentspresented herein.

It is well known by those skilled in the art that it is possible toincrease or decrease the length of an antisense compound and/orintroduce mismatch bases without eliminating activity. For example, inWoolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992,incorporated herein by reference), a series of ASOs 13-25 nucleobases inlength were tested for their ability to induce cleavage of a target RNA.ASOs 25 nucleobases in length with 8 or 11 mismatch bases near the endsof the ASOs were able to direct specific cleavage of the target mRNA,albeit to a lesser extent than the ASOs that contained no mismatches.Similarly, target specific cleavage was achieved using a 13 nucleobaseASOs, including those with 1 or 3 mismatches. Maher and Dolnick (Nuc.Acid. Res. 16:3341-3358, 1988, incorporated herein by reference) testeda series of tandem 14 nucleobase ASOs, and a 28 and 42 nucleobase ASOscomprised of the sequence of two or three of the tandem ASOs,respectively, for their ability to arrest translation of human DHFR in arabbit reticulocyte assay. Each of the three 14 nucleobase ASOs alonewere able to inhibit translation, albeit at a more modest level than the28 or 42 nucleobase ASOs. It is understood that antisense compounds ofthe instant invention can vary in length and percent complementarity tothe target provided that they maintain the desired activity. Methods todetermine desired activity are disclosed herein and well known to thoseskilled in the art.

Target Nucleic Acids

As used herein, “targeting” or “targeted to” refer to the process ofdesigning an oligomeric compound such that the compound specificallyhybridizes with a selected nucleic acid molecule.

“Targeting” an oligomeric compound to a particular target nucleic acidmolecule can be a multistep process. The process usually begins with theidentification of a target nucleic acid whose expression is to bemodulated. As used herein, the terms “target nucleic acid” and “nucleicacid encoding SMN2” encompass DNA encoding SMN2, RNA (including pre-mRNAand mRNA) transcribed from such DNA, and also cDNA derived from suchRNA. For example, the target nucleic acid can be a cellular gene (ormRNA transcribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. As disclosed herein, the target nucleic acid encodesSMN2. In one preferred embodiment, the target nucleic acid is SMN2pre-mRNA.

Target Regions, Segments, and Sites

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect (e.g.,modulation of splicing) will result. “Region” is defined as a portion ofthe target nucleic acid having at least one identifiable structure,function, or characteristic. Target regions may include an exon or anintron. Within regions of target nucleic acids are segments. “Segments”are defined as smaller or sub-portions of regions within a targetnucleic acid. “Sites,” as used in the present invention, are defined asunique nucleobase positions within a target nucleic acid.

Kits, Research Reagents and Diagnostics

The antisense compounds of the present invention can be utilized fordiagnostics, and as research reagents and kits. Furthermore, antisensecompounds, which are able to inhibit gene expression or modulate geneexpression (e.g., modulation of splicing) with specificity, are oftenused by those of ordinary skill to elucidate the function of particulargenes or to distinguish between functions of various members of abiological pathway.

For use in kits and diagnostics, the antisense compounds of the presentinvention, either alone or in combination with other compounds ortherapeutics, can be used as tools in differential and/or combinatorialanalyses to elucidate expression patterns of a portion or the entirecomplement of genes expressed within cells and tissues. Methods of geneexpression analysis are well known to those skilled in the art.

Therapeutics

Antisense compounds of the invention can be used to modulate theexpression of SMN2 in an animal, such as a human. In one non-limitingembodiment, the methods comprise the step of administering to saidanimal in need of therapy for a disease or condition associated withSMN2 an effective amount of an antisense compound that modulatesexpression of SMN2 (e.g. modulates splicing of SMN2). A disease orcondition associated with SMN2 includes, but is not limited to, spinalmuscular atrophy. In one embodiment, the antisense compounds of thepresent invention effectively modulate splicing of SMN2, resulting in anincrease in exon 7 inclusion. Antisense compounds of the presentinvention that effectively modulate expression of SMN2 RNA or proteinproducts of expression are considered active antisense compounds.

For example, modulation of expression of SMN2 can be measured in abodily fluid, which may or may not contain cells; tissue; or organ ofthe animal. Methods of obtaining samples for analysis, such as bodyfluids (e.g., sputum, serum), tissues (e.g., biopsy), or organs, andmethods of preparation of the samples to allow for analysis are wellknown to those skilled in the art. Methods for analysis of RNA andprotein levels are discussed above and are well known to those skilledin the art. The effects of treatment can be assessed by measuringbiomarkers associated with the target gene expression in theaforementioned fluids, tissues or organs, collected from an animalcontacted with one or more compounds of the invention, by routineclinical methods known in the art. These biomarkers include but are notlimited to: liver transaminases, bilirubin, albumin, blood ureanitrogen, creatine and other markers of kidney and liver function;interleukins, tumor necrosis factors, intracellular adhesion molecules,C-reactive protein, chemokines, cytokines, and other markers ofinflammation.

The antisense compounds of the present invention can be utilized inpharmaceutical compositions by adding an effective amount of a compoundto a suitable pharmaceutically acceptable diluent or carrier. Acceptablecarriers and diluents are well known to those skilled in the art.Selection of a diluent or carrier is based on a number of factors,including, but not limited to, the solubility of the compound and theroute of administration. Such considerations are well understood bythose skilled in the art. In one aspect, the antisense compounds of thepresent invention modulate splicing of SMN2. The compounds of theinvention can also be used in the manufacture of a medicament for thetreatment of diseases and disorders related to SMN2.

Methods whereby bodily fluids, organs or tissues are contacted with aneffective amount of one or more of the antisense compounds orcompositions of the invention are also contemplated. Bodily fluids,organs or tissues can be contacted with one or more of the compounds ofthe invention resulting in modulation of SMN2 expression in the cells ofbodily fluids, organs or tissues. An effective amount can be determinedby monitoring the modulatory effect of the antisense compound orcompounds or compositions on target nucleic acids or their products bymethods routine to the skilled artisan.

Thus, provided herein is the use of an isolated antisense compoundtargeted to SMN2 in the manufacture of a medicament for the treatment ofa disease or disorder by means of the method described above. In oneembodiment, the antisense compound is targeted to exon 7 of SMN2. Inanother embodiment, the antisense compound is targeted to intron 6 ofSMN2. In yet another embodiment, the antisense compound is targeted tointron 7 of SMN2.

Chemical Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base(sometimes referred to as a “nucleobase” or simply a “base”). The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety ofthe sugar. In forming oligonucleotides, the phosphate groups covalentlylink adjacent nucleosides to one another to form a linear polymericcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage. It is often preferable to include chemicalmodifications in oligonucleotides to alter their activity. Chemicalmodifications can alter oligonucleotide activity by, for example:increasing affinity of an antisense oligonucleotide for its target RNA,increasing nuclease resistance, and/or altering the pharmacokinetics ofthe oligonucleotide. The use of chemistries that increase the affinityof an oligonucleotide for its target can allow for the use of shorteroligonucleotide compounds.

The term “nucleobase” or “heterocyclic base moiety” as used herein,refers to the heterocyclic base portion of a nucleoside. In general, anucleobase is any group that contains one or more atom or groups ofatoms capable of hydrogen bonding to a base of another nucleic acid. Inaddition to “unmodified” or “natural” nucleobases such as the purinenucleobases adenine (A) and guanine (G), and the pyrimidine nucleobasesthymine (T), cytosine (C) and uracil (U), many modified nucleobases ornucleobase mimetics known to those skilled in the art are amenable tothe present invention. The terms modified nucleobase and nucleobasemimetic can overlap but generally a modified nucleobase refers to anucleobase that is fairly similar in structure to the parent nucleobase,such as for example a 7-deaza purine, a 5-methyl cytosine, or a G-clamp,whereas a nucleobase mimetic would include more complicated structures,such as for example a tricyclic phenoxazine nucleobase mimetic. Methodsfor preparation of the above noted modified nucleobases are well knownto those skilled in the art.

Antisense compounds of the present invention may also contain one ormore nucleosides having modified sugar moieties. The furanosyl sugarring of a nucleoside can be modified in a number of ways including, butnot limited to, addition of a substituent group, bridging of twonon-geminal ring atoms to form a bicyclic nucleic acid (BNA) andsubstitution of an atom or group such as —S—, —N(R)— or —C(R₁)(R₂) forthe ring oxygen at the 4′-position. Modified sugar moieties are wellknown and can be used to alter, typically increase, the affinity of theantisense compound for its target and/or increase nuclease resistance. Arepresentative list of preferred modified sugars includes but is notlimited to bicyclic modified sugars (BNA's), including LNA and ENA(4′-(CH₂)₂—O-2′ bridge); and substituted sugars, especially2′-substituted sugars having a 2′-F, 2′-OCH₂ or a 2′-O(CH₂)₂—OCH₃substituent group. Sugars can also be replaced with sugar mimetic groupsamong others. Methods for the preparations of modified sugars are wellknown to those skilled in the art.

The present invention includes internucleoside linking groups that linkthe nucleosides or otherwise modified monomer units together therebyforming an antisense compound. The two main classes of internucleosidelinking groups are defined by the presence or absence of a phosphorusatom. Representative phosphorus containing internucleoside linkagesinclude, but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates.Representative non-phosphorus containing internucleoside linking groupsinclude, but are not limited to, methylenemethylimino(—CH₂—N(CH₃)—O—CH₂—), thiodiester (—O—C(O)—S—), thionocarbamate(—O—C(O)(NH)—S—); siloxane (—O—Si(H)2-O—); and N,N′-dimethylhydrazine(—CH₂—N(CH₃)—N(CH₃)—). Antisense compounds having non-phosphorusinternucleoside linking groups are referred to as oligonucleosides.Modified internucleoside linkages, compared to natural phosphodiesterlinkages, can be used to alter, typically increase, nuclease resistanceof the antisense compound. Internucleoside linkages having a chiral atomcan be prepared racemic, chiral, or as a mixture. Representative chiralinternucleoside linkages include, but are not limited to,alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing linkages are wellknown to those skilled in the art.

As used herein the term “mimetic” refers to groups that are substitutedfor a sugar, a nucleobase, and/or internucleoside linkage. Generally, amimetic is used in place of the sugar or sugar-internucleoside linkagecombination, and the nucleobase is maintained for hybridization to aselected target. Representative examples of a sugar mimetic include, butare not limited to, cyclohexenyl or morpholino. Representative examplesof a mimetic for a sugar-internucleoside linkage combination include,but are not limited to, peptide nucleic acids (PNA) and morpholinogroups linked by uncharged achiral linkages. In some instances a mimeticis used in place of the nucleobase. Representative nucleobase mimeticsare well known in the art and include, but are not limited to, tricyclicphenoxazine analogs and universal bases (Berger et al., Nuc Acid Res.2000, 28:2911-14, incorporated herein by reference). Methods ofsynthesis of sugar, nucleoside and nucleobase mimetics are well known tothose skilled in the art.

As used herein the term “nucleoside” includes, nucleosides, abasicnucleosides, modified nucleosides, and nucleosides having mimetic basesand/or sugar groups.

In the context of this invention, the term “oligonucleotide” refers toan oligomeric compound which is an oligomer or polymer of ribonucleicacid (RNA) or deoxyribonucleic acid (DNA). This term includesoligonucleotides composed of naturally- and non-naturally-occurringnucleobases, sugars and covalent internucleoside linkages, possiblyfurther including non-nucleic acid conjugates.

The present invention provides compounds having reactive phosphorusgroups useful for forming internucleoside linkages including for examplephosphodiester and phosphorothioate internucleoside linkages. Methods ofpreparation and/or purification of precursors or antisense compounds ofthe instant invention are not a limitation of the compositions ormethods of the invention. Methods for synthesis and purification of DNA,RNA, and the antisense compounds of the instant invention are well knownto those skilled in the art.

As used herein the term “chimeric antisense compound” refers to anantisense compound, having at least one sugar, nucleobase and/orinternucleoside linkage that is differentially modified as compared tothe other sugars, nucleobases and internucleoside linkages within thesame oligomeric compound. The remainder of the sugars, nucleobases andinternucleoside linkages can be independently modified or unmodified. Ingeneral a chimeric oligomeric compound will have modified nucleosidesthat can be in isolated positions or grouped together in regions thatwill define a particular motif. Any combination of modifications and ormimetic groups can comprise a chimeric oligomeric compound of thepresent invention.

Chimeric oligomeric compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligomeric compound mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease thatcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of inhibition of gene expression. Consequently,comparable results can often be obtained with shorter oligomericcompounds when chimeras are used, compared to for examplephosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

As used in the present invention the term “fully modified motif” refersto an antisense compound comprising a contiguous sequence of nucleosideswherein essentially each nucleoside is a sugar modified nucleosidehaving uniform modification.

The compounds described herein contain one or more asymmetric centersand thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), a or B, or as (D) or (L) such as foramino acids et al. The present invention is meant to include all suchpossible isomers, as well as their racemic and optically pure forms.

In one aspect of the present invention antisense compounds are modifiedby covalent attachment of one or more conjugate groups. Conjugate groupsmay be attached by reversible or irreversible attachments. Conjugategroups may be attached directly to anti sense compounds or by use of alinker. Linkers may be mono- or bifunctional linkers. Such attachmentmethods and linkers are well known to those skilled in the art. Ingeneral, conjugate groups are attached to antisense compounds to modifyone or more properties. Such considerations are well known to thoseskilled in the art.

Oligomer Synthesis

Oligomerization of modified and unmodified nucleosides can be routinelyperformed according to literature procedures for DNA (Protocols forOligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/orRNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications ofChemically synthesized RNA in RNA: Protein Interactions, Ed. Smith(1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).

Antisense compounds of the present invention can be conveniently androutinely made through the well-known technique of solid phasesynthesis. Equipment for such synthesis is sold by several vendorsincluding, for example, Applied Biosystems (Foster City, Calif.). Anyother means for such synthesis known in the art may additionally oralternatively be employed. It is well known to use similar techniques toprepare oligonucleotides such as the phosphorothioates and alkylatedderivatives. The invention is not limited by the method of antisensecompound synthesis.

Oligomer Purification and Analysis

Methods of oligonucleotide purification and analysis are known to thoseskilled in the art. Analysis methods include capillary electrophoresis(CE) and electrospray-mass spectroscopy. Such synthesis and analysismethods can be performed in multi-well plates. The method of theinvention is not limited by the method of oligomer purification.

Salts, Prodrugs and Bioequivalents

The antisense compounds of the present invention comprise anypharmaceutically acceptable salts, esters, or salts of such esters, orany other functional chemical equivalent which, upon administration toan animal including a human, is capable of providing (directly orindirectly) the biologically active metabolite or residue thereof.Accordingly, for example, the disclosure is also drawn to prodrugs andpharmaceutically acceptable salts of the antisense compounds of thepresent invention, pharmaceutically acceptable salts of such prodrugs,and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive or less active form that is converted to an active form (i.e.,drug) within the body or cells thereof by the action of endogenousenzymes, chemicals, and/or conditions. In particular, prodrug versionsof the oligonucleotides of the invention are prepared as SATE((S-acetyl-2-thioethyl) phosphate) derivatives according to the methodsdisclosed in WO 93/24510 or WO 94/26764. Prodrugs can also includeantisense compounds wherein one or both ends comprise nucleobases thatare cleaved (e.g., by incorporating phosphodiester backbone linkages atthe ends) to produce the active compound.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.Sodium salts of antisense oligonucleotides are useful and are wellaccepted for therapeutic administration to humans. In anotherembodiment, sodium salts of dsRNA compounds are also provided.

Formulations

The antisense compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. In a preferred embodiment,administration is topical to the surface of the respiratory tract,particularly pulmonary, e.g., by nebulization, inhalation, orinsufflation of powders or aerosols, by mouth and/or nose.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers,finely divided solid carriers, or both, and then, if necessary, shapingthe product (e.g., into a specific particle size for delivery). In apreferred embodiment, the pharmaceutical formulations of the instantinvention are prepared for pulmonary administration in an appropriatesolvent, e.g., water or normal saline, possibly in a sterileformulation, with carriers or other agents to allow for the formation ofdroplets of the desired diameter for delivery using inhalers, nasaldelivery devices, nebulizers, and other devices for pulmonary delivery.Alternatively, the pharmaceutical formulations of the instant inventionmay be formulated as dry powders for use in dry powder inhalers.

A “pharmaceutical carrier” or “excipient” can be a pharmaceuticallyacceptable solvent, suspending agent or any other pharmacologicallyinert vehicle for delivering one or more nucleic acids to an animal andare known in the art. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition.

Combinations

Compositions of the invention can contain two or more antisensecompounds. In another related embodiment, compositions of the presentinvention can contain one or more antisense compounds, particularlyoligonucleotides, targeted to a first nucleic acid and one or moreadditional antisense compounds targeted to a second nucleic acid target.Alternatively, compositions of the present invention can contain two ormore antisense compounds targeted to different regions of the samenucleic acid target. Two or more combined compounds may be used togetheror sequentially. Compositions of the instant invention can also becombined with other non-antisense compound therapeutic agents.

NONLIMITING DISCLOSURE AND INCORPORATION BY REFERENCE

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate thecompounds of the invention and are not intended to limit the same. Eachof the references, GenBank accession numbers, and the like recited inthe present application is incorporated herein by reference in itsentirety.

Example 1 Design of Modified Antisense Compounds Targeting SMN2

In accordance with the present invention, antisense compounds weredesigned to target intron 6, exon 7 or intron 7 of SMN2 (SEQ ID NO: 1).In reference to SEQ ID NO:1, nucleotides 61-114 represent exon 7, whilenucleotides 1-60 and 115-174 represent portions of intron 6 and intron7, respectively. The compounds, listed in Table 1, are either 12, 15, 16or 18 nucleotides in length and are composed of 2′-O-methoxyethylnucleotides, also known as 2′-MOE nucleotides. The internucleoside(backbone) linkages are phosphodiester throughout the oligonucleotide.All cytidine residues are 5-methylcytidines. Target site indicates thefirst (5′-most) nucleotide number of the target sequence (SEQ ID NO: 1)to which the oligonucleotide binds.

TABLE 1 2′-MOE Compounds Targeting SMN2 SEQ ISIS Target Target SequenceID # Site Region Length (5′ to 3′) NO 390645   1 Intron 6 15TAGATAGCTATATAT   2 393593   2 Intron 6 15 ATAGATAGCTATATA   3 393592  3 Intron 6 15 TATAGATAGCTATAT   4 393591   4 Intron 6 15ATATAGATAGCTATA   5 393590   5 Intron 6 15 GATATAGATAGCTAT   6 393602  5 Intron 6 12 ATAGATAGCTAT   7 390644   6 Intron 6 15 AGATATAGATAGCTA  8 393601   6 Intron 6 12 TATAGATAGCTA   9 393589   7 Intron 6 15TAGATATAGATAGCT  10 393600   7 Intron 6 12 ATATAGATAGCT  11 393588   8Intron 6 15 ATAGATATAGATAGC  12 393599   8 Intron 6 12 GATATAGATAGC  13393587   9 Intron 6 15 TATAGATATAGATAG  14 393598   9 Intron 6 12AGATATAGATAG  15 393586  10 Intron 6 15 ATATAGATATAGATA  16 393597  10Intron 6 12 TAGATATAGATA  17 390643  11 Intron 6 15 TATATAGATATAGAT  18393596  11 Intron 6 12 ATAGATATAGAT  19 393595  12 Intron 6 12TATAGATATAGA  20 393594  13 Intron 6 12 ATATAGATATAG  21 390642  16Intron 6 15 ATAGCTATATAGATA  22 390641  21 Intron 6 15 AAAAAATAGCTATAT 23 390640  26 Intron 6 15 GTTAAAAAAAATAGC  24 390639  31 Intron 6 15AGGAAGTTAAAAAAA  25 390638  36 Intron 6 15 AATAAAGGAAGTTAA  26 390637 41 Intron 6 15 AGGAAAATAAAGGAA  27 390636  46 Intron 6 15CTGTAAGGAAAATAA  28 372641  61 Exon 7 15 ATTTTGTCTAAAACC  29 385909  62Exon 7 15 GATTTTGTCTAAAAC  30 383497  63 Exon 7 12 TTTTGTCTAAAA  31385908  63 Exon 7 15 TGATTTTGTCTAAAA  32 383496  64 Exon 7 12ATTTTGTCTAAA  33 385907  64 Exon 7 15 TTGATTTTGTCTAAA  34 383495  65Exon 7 12 GATTTTGTCTAA  35 385906  65 Exon 7 15 TTTGATTTTGTCTAA  36385910  65 Exon 7 16 TTTTGATTTTGTCTA  37 A 372642  66 Exon 7 15TTTTGATTTTGTCTA  38 383494  66 Exon 7 12 TGATTTTGTCTA  39 383493  67Exon 7 12 TTGATTTTGTCT  40 385905  67 Exon 7 15 TTTTTGATTTTGTCT  41383492  68 Exon 7 12 TTTGATTTTGTC  42 385904  68 Exon 7 15CTTTTTGATTTTGTC  43 383491  69 Exon 7 12 TTTTGATTTTGT  44 383490  70Exon 7 12 TTTTTGATTTTG  45 372643  71 Exon 7 15 CTTCTTTTTGATTTT  46383489  71 Exon 7 12 CTTTTTGATTTT  47 383488  72 Exon 7 12 TCTTTTTGATTT 48 372644  76 Exon 7 15 CCTTCCTTCTTTTTG  49 372645  81 Exon 7 15GAGCACCTTCCTTCT  50 372646  86 Exon 7 15 AATGTGAGCACCTTC  51 372647  91Exon 7 15 TAAGGAATGTGAGCA  52 383470  92 Exon 7 18 AATTTAAGGAATGTG  53AGC 383477  92 Exon 7 15 TTAAGGAATGTGAGC  54 383469  93 Exon 7 18TAATTTAAGGAATGT  55 GAG 383476  93 Exon 7 15 TTTAAGGAATGTGAG  56 383487 93 Exon 7 12 AAGGAATGTGAG  57 383468  94 Exon 7 18 TTAATTTAAGGAATG  58TGA 383475  94 Exon 7 15 ATTTAAGGAATGTGA  59 383486  94 Exon 7 12TAAGGAATGTGA  60 383467  95 Exon 7 18 CTTAATTTAAGGAAT  61 GTG 383474  95Exon 7 15 AATTTAAGGAATGTG  62 383485  95 Exon 7 12 TTAAGGAATGTG  63372648  96 Exon 7 15 TAATTTAAGGAATGT  64 383466  96 Exon 7 18CCTTAATTTAAGGAA  65 TGT 383484  96 Exon 7 12 TTTAAGGAATGT  66 383473  97Exon 7 15 TTAATTTAAGGAATG  67 383483  97 Exon 7 12 ATTTAAGGAATG  68383472  98 Exon 7 15 CTTAATTTAAGGAAT  69 383482  98 Exon 7 12AATTTAAGGAAT  70 383471  99 Exon 7 15 CCTTAATTTAAGGAA  71 383481  99Exon 7 12 TAATTTAAGGAA  72 372649 100 Exon 7 15 TCCTTAATTTAAGGA  73383480 100 Exon 7 12 TTAATTTAAGGA  74 383479 101 Exon 7 12 CTTAATTTAAGG 75 383478 102 Exon 7 12 CCTTAATTTAAG  76 390646 115 Intron 7 15TGCTGGCAGACTTAC  77 390647 120 Intron 7 15 CATAATGCTGGCAGA  78 393610121 Intron 7 15 TCATAATGCTGGCAG  79 393609 122 Intron 7 15TTCATAATGCTGGCA  80 393608 123 Intron 7 15 TTTCATAATGCTGGC  81 387949124 Intron 7 20 ATTCACTTTCATAAT  82 GCTGG 393607 124 Intron 7 15CTTTCATAATGCTGG  83 393619 124 Intron 7 12 TCATAATGCTGG  84 390648 125Intron 7 15 ACTTTCATAATGCTG  85 393618 125 Intron 7 12 TTCATAATGCTG  86393606 126 Intron 7 15 CACTTTCATAATGCT  87 393617 126 Intron 7 12TTTCATAATGCT  88 393605 127 Intron 7 15 TCACTTTCATAATGC  89 393616 127Intron 7 12 CTTTCATAATGC  90 393604 128 Intron 7 15 TTCACTTTCATAATG  91393615 128 Intron 7 12 ACTTTCATAATG  92 393603 129 Intron 7 15ATTCACTTTCATAAT  93 393614 129 Intron 7 12 CACTTTCATAAT  94 390649 130Intron 7 15 GATTCACTTTCATAA  95 393613 130 Intron 7 12 TCACTTTCATAA  96393612 131 Intron 7 12 TTCACTTTCATA  97 393611 132 Intron 7 12ATTCACTTTCAT  98 390650 135 Intron 7 15 AGTAAGATTCACTTT  99 390651 140Intron 7 15 ACAAAAGTAAGATTC 100 390652 145 Intron 7 15 GTTTTACAAAAGTAA101 390653 150 Intron 7 15 ATAAAGTTTTACAAA 102 390654 155 Intron 7 15AAACCATAAAGTTTT 103 390655 160 Intron 7 15 TCCACAAACCATAAA 104

Other nucleic acid sequences for SMN genes are publicly available andwell known in the art. For example, Genbank Accession Nos. NM_000344,NM_022874, NM_022875, U43883, AC140134, AC139778, AC010237, AC022119 andAC004999 provide nucleotide sequences of SMN1 or SMN2.

Example 2

Treatment with Oligomeric Compounds

When cells reach appropriate confluency, they are treated witholigonucleotide using a transfection method as described.

LIPOFECTIN™

When cells reach 65-75% confluency, they are treated witholigonucleotide. Oligonucleotide is mixed with LIPOFECTIN™ InvitrogenLife Technologies, Carlsbad, Calif.) in Opti-MEM™-1 reduced serum medium(Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desiredconcentration of oligonucleotide and a LIPOFECTIN™ concentration of 2.5or 3 μg/mL per 100 nM oligonucleotide. This transfection mixture isincubated at room temperature for approximately 0.5 hours. For cellsgrown in 96-well plates, wells are washed once with 100 μL OPTI-MEM™-1and then treated with 130 μL of the transfection mixture. Cells grown in24-well plates or other standard tissue culture plates are treatedsimilarly, using appropriate volumes of medium and oligonucleotide.Cells are treated and data are obtained in duplicate or triplicate.After approximately 4-7 hours of treatment at 37° C., the mediumcontaining the transfection mixture is replaced with fresh culturemedium.

Electroporation

When cells reach approximately 80% confluency, oligonucleotide isintroduced via electroporation. Oligonucleotide concentrations used inelectroporation experiments range from 0.1 to 40 μM. Cells are harvestedby routine trypsinization to produce a single cell suspension. Followingcell counting using a hemocytometer and pelleting by centrifugation,cells are resuspended in OPTI-MEM™-1 reduced serum medium (InvitrogenLife Technologies, Carlsbad, Calif.) to achieve a density of 1×10⁷cells/mL. Cells are mixed with the desired concentration ofoligonucleotide and transferred to a 0.1 cm electroporation cuvette (BTXMolecular Delivery Systems, Hollister, Mass.). Cells are subjected to asingle pulse using an electroporation apparatus (for example, the BTXElectro Square Porator T820 or the BTX HT300, BTX Molecular DeliverySystems, Hollister, Mass.), diluted into culture medium and plated into24-well plates. Cells are treated and data were obtained in duplicate ortriplicate.

Example 3 Minigenes for SMN2 Splicing Studies

All SMN constructs are derivatives of pCITel (Lorson and Androphy, Hum.Mol. Genet., 2000, 9, 259-265). The primers used to generate each SMN2construct are shown in Table 2. Using a Quickchange kit (Stratagene, LaJolla, Calif.), an XbaI site was inserted by site-directed mutagenesisat nucleotide 7170 (in intron 7) to generate pCI-SMNx-wt. For in vitrotranscription studies, intron 6 was shortened by overlap-extension PCRto generate pCISMNxΔ6-wt, deleting 5,570 nt from position 1235 to theBclI site at nt 6805. Two sets of PCR were performed with Pfu polymeraseand pCISMNx-wt as template. The first PCR was carried out with primersCIF1 and Δ6-bclR, the second with primers smnΔ6-vrlp and CIR. The PCRproducts were purified, combined and reamplified with the outer primers(CIF1 and CIR). The final product was digested with XhoI and NotI andsubcloned it into pCISMNx-wt digested with the same enzymes. All theconstructs were verified by direct sequencing. Templates were generatedfor in vitro transcription by PCR amplification of pCISMNxΔ6-wt usingprimers CIF2 and smn8-75+5R. The final products contained a T7 promoter,exon 6 (124 nt), a shortened intron 6 (200 nt), exon 7 (54 nt), intron 7(444 nt), and 75 nt of exon 8 followed by a consensus 5′ splice site(GTAAGTACTT; SEQ ID NO: 22) (Cartegni and Krainer, Nature Genet., 2002,30, 377-384; WO 02/38738).

TABLE 2 Primers used to generate SMN2 minigenes and templates SEQ PrimerID Construct Name Primer Sequence NO pCI-SMNx-wt smnI7xbaFAGATAAAAGGTTAATCTAGATCCC 106 TACTAGAATTCTC pCI-SMNx-wt smnI7xbaRGAGAATTCTAGTAGGGATCTAGAT 107 TAACCTTTTATCT pCISMNxΔ6-wt CIF1AATTGCTAACGCAGTCAGTGCTTC 108 pCISMNxΔ6-wt Δ6-bclRAATATGATCAGCAAAACAAAGTCA 109 CATAACTAC pCISMNxΔ6-wt smnΔ6-GTGACTTTGTTTTGCTGATCATAT 110 vrlp TTTGTTGAATAAAATAAG pCISMNxΔ6-wt CIRAATGTATCTTATCATGTCTGCTCG 111 In vitro CIF2 AATGTATCTTATCATGTCTGCTCG 112templates In vitro Smn8-75 + AAGTACTTACCTGTAACGCTTCAC 113 templates 5′RATTCCAGATCTGTC

Example 4 Effect of Antisense Compounds on SMN2 Splicing in Cell-FreeExtracts

2′-MOE antisense compounds designed to target exon 7 of SMN2 wereevaluated for their effect on splicing of SMN2. Templates for in vitroSMN2 splicing studies were generated as described in Example 3.5′-capped T7 runoff transcripts from purified PCR products wereuniformly labeled with [α-³²P]-UTP and purified by denaturingpolyacrylamide gel electrophoresis. Labeled in vitro transcripts werespliced in HeLa cell nuclear or S100 extracts (Mayeda and Krainer,Methods Mol. Biol., 1999, 118, 315-321; Mayeda and Krainer, Methods Mol.Biol., 1999, 118, 309-314) by incubating 10 fmol of transcript in 12.5μl standard splicing reactions containing 3 μl of nuclear extract or 2μl of S100 extract. Extracts either contained no antisenseoligonucleotide or were complemented with 1, 5, 10, 25, 50, 100, 200 or400 nM of ISIS 372641, ISIS 372642, ISIS 372643, ISIS 372644, ISIS372645, ISIS 372646, ISIS 372647, ISIS 372648 or ISIS 372649. Controloligonucleotide ISIS 372693 was also used in this study(TTGTATTCTATGTTT; SEQ ID NO: 114). The MgCl₂ concentration of thesplicing mixture was 1.6 mM. After incubation at 30° C. for 4 h, RNA wasextracted and analyzed on 8% denaturing polyacrylamide gels, followed byautoradiography and phosphorimager analysis. Exon inclusion wascalculated as a percentage of the total amount of spliced mRNA (includedmRNA×100/(included mRNA+skipped mRNA).

The results showed that several of the SMN2 antisense oligonucleotidesaltered splicing of SMN2 exon 7, while control oligonucleotide ISIS372693 had no effect. ISIS 372641 promoted skipping of SMN2 exon 7 in adose-dependent manner. Exon 7 was included in only 2% of SMN2 splicedtranscripts incubated with 400 nM of ISIS 372641, compared with 26% oftranscripts incubated with no oligonucleotide. Similarly, ISIS 372646inhibited inclusion of exon 7 in a dose-dependent manner with 16% ofSMN2 spliced transcripts containing exon 7, compared with 32% oftranscripts incubated without oligonucleotide. In contrast, ISIS 372642inhibited skipping of exon 7 in a dose-dependent manner. The percentageof SMN2 spliced transcripts containing exon 7 increased from 28% whenincubated without oligonucleotide to 40% when incubated with 400 nM ofISIS 372642. ISIS 372648 also increased inclusion of exon 7 with 69% ofSMN2 transcripts containing exon 7 when incubated with the highestconcentration of oligonucleotide, compared with 42% of transcripts whenincubated without oligonucleotide. Extracts containing ISIS 372643 alsoshowed a slight increase in exon 7 inclusion at the higheroligonucleotide concentrations. Taken together, these results illustratethat antisense oligonucleotides targeting exon 7 of SMN2 are capable ofaltering splicing of transcripts to either promote or inhibit inclusionof exon 7.

Example 5 Effect of Antisense Compounds on SMN2 Splicing in HEK293 Cells

Antisense compounds targeting SMN2 exon 7 were evaluated for theireffects on SMN2 splicing in cultured cells. HEK293 cells wereelectroporated with 10 μg SMN2 minigene and 10 μM of either SMN2antisense oligonucleotide ISIS 372641, ISIS 372642, ISIS 372643, ISIS372644, ISIS 372645, ISIS 372646, ISIS 372647, ISIS 372648 or ISIS372649, or control oligonucleotide ISIS 372693. Sixty hours aftertransfection, total RNA was isolated using Trizol Reagent (Invitrogen,Carlsbad, Calif.) following the manufacturer's directions. One μg ofDNAse-treated total RNA was used to generate first-strand cDNA sequenceswith oligo(dT) and Superscript II reverse transcriptase (Invitrogen),and the cDNA was amplified semi-quantitatively by 16 PCR cycles (94° C.for 30 s, 57.5° C. for 30 s, 72° C. for 90 s) in the presence of[α-³²P]dCTP (Lorson and Androphy, Hum. Mol. Genet., 2000, 9, 259-265).PCR products were analyzed by electrophoresis on 6% denaturingpolyacrylamide gels, followed by autoradiography and phosphorimageranalysis. Exon inclusion was calculated as a percentage of the totalamount of spliced mRNA (included mRNA×100/(included mRNA+skipped mRNA).The percentage of SMN2 spliced transcripts containing exon 7 (%inclusion) is shown in Table 3. The target site of each oligonucleotiderelative to SEQ ID NO: 1 is also indicated.

TABLE 3 Effect of SMN2 antisense compounds on exon 7 inclusion TargetISIS # Site % Inclusion 372641 61 6.4 372642 66 67.4 372643 71 34.9372644 76 12.9 372645 81 7.8 372646 86 11.8 372647 91 9.5 372648 96 75.2372649 100 55.1 372693 Control 57.7

Compared to control oligonucleotide, transfection with either ISIS372642 or 372648 resulted in a greater percentage of SMN2 transcriptswith exon 7 included, which is consistent with results obtained from invitro assays. Treatment with ISIS 372641, ISIS 372644, ISIS 372645, ISIS372646 and ISIS 372647 resulted in the most significant increase in exon7 skipping.

SMN2 antisense oligonucleotides were further evaluated for their effectson endogenous SMN1 and SMN2 pre-mRNA splicing in cultured cells. HEK293cells were electroporated with 10 μM of either SMN2 antisenseoligonucleotide ISIS 372641, ISIS 372642, ISIS 372643, ISIS 372644, ISIS372645, ISIS 372646, ISIS 372647, ISIS 372648 or ISIS 372649, or controloligonucleotide ISIS 372693. Sixty hours after transfection, RNA wasisolated and RT-PCR was performed as described above to examine splicingchanges of both SMN1 and SMN2 pre-mRNAs. PCR products were digested withDdeI to distinguish between SMN1 and SMN2, separated by electrophoresison 6% denaturing polyacrylamide gels and analyzed by autoradiography.The percentage of SMN1 and SMN2 spliced transcripts containing exon 7 (%inclusion) is shown in Table 4.

TABLE 4 Effect of SMN2 antisense oligonucleotides on SMN1 and SMN2pre-mRNA splicing % Inclusion % Inclusion ISIS # SMN1 SMN2 372641 82.511.1 372642 96.2 69.5 372643 94.1 28.8 372644 68.5 23.8 372645 47.3 15.2372646 57.7 20.2 372647 58.8 12.8 372648 93.1 52.2 372649 94.8 49.3372693 95.1 50.1

In accordance with previous results, transfection with ISIS 372642 andISIS 372648 led to the greatest level of exon 7 inclusion in SMN2pre-mRNA transcripts. ISIS 372641, ISIS 372644, ISIS 372645, ISIS 372646and ISIS 372647 significantly reduced the percentage of SMN1 transcriptscontaining exon 7. These oligonucleotides, along with ISIS 372643, alsoreduced exon 7 inclusion in SMN2 mRNAs.

Additional antisense oligonucleotides targeting the 3′ end of SMN2 exon7 (see Table 1) were evaluated for their effects on SMN2 pre-mRNAsplicing. HEK293 cells were electroporated with 10 μM of either SMN2antisense oligonucleotide ISIS 383466, ISIS 383467, ISIS 383468, ISIS383469, ISIS 383470, ISIS 383471, ISIS 383472, ISIS 383473, ISIS 383474,ISIS 383475, ISIS 383476, ISIS 383477 or ISIS 372648, or a controloligonucleotide. Fifty hours after transfection, RNA was isolated andRT-PCR was performed as described above to examine splicing changes ofSMN2 pre-mRNA. PCR products were digested with DdeI to distinguishbetween SMN1 and SMN2, separated by electrophoresis on 6% denaturingpolyacrylamide gels and analyzed by autoradiography. The percentage ofSMN2 spliced transcripts containing exon 7 (% inclusion) is shown inTable 5. The length and target site of each oligonucleotide relative toSEQ ID NO: 1 are also indicated.

TABLE 5 Effect of SMN2 antisense oligonucleotides on SMN2 pre-mRNAsplicing Target ISIS # Site Length % Inclusion 383470 92 18 4.9 38347792 15 5.3 383469 93 18 18.9 383476 93 15 32.8 383468 94 18 18.7 38347594 15 84.8 383467 95 18 8.1 383474 95 15 77.0 372648 96 15 59.6 38346696 18 37.5 383473 97 15 42.2 383472 98 15 45.0 383471 99 15 37.1 ControlN/A N/A 41.3 Vehicle N/A N/A 41.4

The results demonstrate that a number of SMN2 antisense oligonucleotidescan alter splicing of SMN2 pre-mRNAs. ISIS 383467, ISIS 383468, ISIS383469, ISIS 383470, ISIS 383476 and ISIS 383477 inhibited inclusion ofexon 7; ISIS 383474, ISIS 383475 and ISIS 372648 significantly increasedinclusion of exon 7; and ISIS 383466, ISIS 383471, ISIS 383472 and ISIS383473 appeared to have little effect on SMN2 splicing, relative tooligonucleotide and vehicle controls. These results suggest that SMN2oligonucleotides with a target site between nucleotides 94-96 areparticularly effective at achieving inclusion of exon 7 during SMN2pre-mRNA splicing, and further suggests oligonucleotides 15 nucleotidesin length are more effective than those 18 nucleotides in length.

Example 6 Effect of Antisense Compounds on SMN2 Splicing in SMAFibroblast Cells

In accordance with the present invention, SMN2 antisenseoligonucleotides were tested in fibroblast cells derived from a patientwith type I SMA (3813 cell line; Coovert et al., Human Mol. Genet.,1997, 6, 1205-1214). SMA fibroblasts contain SMN2, but do not expressSMN1. SMA fibroblasts were lipofected with 200 nM of either SMN2antisense oligonucleotide ISIS 372641, ISIS 372642, ISIS 372643, ISIS372644, ISIS 372645, ISIS 372646, ISIS 372647, ISIS 372648 or ISIS372649, or control oligonucleotide ISIS 372693. Seventy hours aftertransfection, RNA was isolated and RT-PCR was performed as describedabove to examine splicing changes of endogenous SMN2 pre-mRNAs. PCRproducts were separated by electrophoresis and analyzed byautoradiography. The percentage of SMN2 spliced transcripts containingexon 7 (% inclusion) is shown in Table 6. The target site of eacholigonucleotide relative to SEQ ID NO: 1 is also indicated.

TABLE 6 Effect on SMN2 antisense oligonucleotides on exon 7 inclusion inSMA fibroblasts Target ISIS # Site % Inclusion 372641 61 41.8 372642 6655.2 372643 71 40.9 372644 76 43.4 372645 81 43.7 372646 86 38.8 37264791 43.6 372648 96 49.8 372649 100 48.8 372693 Control 48.7 PBS N/A 48.8

In accordance with previous findings, treatment with ISIS 372642 andISIS 372648 generated a greater percentage of SMN2 splicing productscontaining exon 7.

A second experiment to further evaluate ISIS 372642 and ISIS 383475 inSMA fibroblasts was performed. SMA fibroblasts were lipofected witheither 200 nM ISIS 372642, 200 nM ISIS 383475, or 100 nM ISIS 372642 incombination with 100 nM ISIS 383475. ISIS 372693 (200 nM) and vehicleonly were also used as controls. Fifty hours after transfection, RNA wasisolated and RT-PCR was performed. PCR products were separated byelectrophoresis and analyzed by autoradiography. The percentage of SMN2spliced transcripts containing exon 7 (% inclusion) is shown in Table 7.

TABLE 7 Effect of ISIS 372642 and ISIS 383475 on exon 7 inclusion in SMAfibroblasts Treatment (ISIS #) % Inclusion 372642 47.8 383475 53.9372642 & 383475 49.2 372693 36.3 Vehicle 35.0

The results demonstrate that treatment with ISIS 372642 or ISIS 383475,either alone or in combination, leads to greater inclusion of exon 7 inSMA transcripts.

Example 7 Microwalk of ISIS 372642 and ISIS 372648 Target Sites

The studies shown above demonstrated that both ISIS 372642 and ISIS372648 were effective in promoting SMN2 exon 7 inclusion. To furtherevaluate the target sites surrounding these compounds, additionalcompounds were designed as 1 nucleotide microwalks around each site (seeTable 1 for sequences and target sites). Ten compounds 12 nucleotides inlength were designed for each microwalk. Seven additional compounds 15or 16 nucleotides in length were designed to target the region of ISIS372642. The antisense compounds targeting the 3′ end of exon 7 (ISIS383466-382477), described above in Example 5, were included forcomparison with the ISIS 372648 microwalk compounds. Each compound wasevaluated in the SMN2 minigene splicing assay and the endogenousSMN1/SMN2 splicing assay in HEK293 cells. Both assays are described inprevious examples herein. The results are in shown in Tables 8 and 9.

TABLE 8 ISIS 372642 Microwalk Compounds: Effect on Exon 7 Inclusion %Inclusion % Inclusion % Inclusion Target SMN2 Endogenous Endogenous ISIS# Site Length Minigene SMN2 SMN1 385909 62 15 44 43 81 383497 63 12 5551 86 385908 63 15 49 53 85 383496 64 12 32 36 81 385907 64 15 56 54 86383495 65 12 54 49 85 385906 65 15 56 57 87 385910 65 16 60 66 89 37264266 15 66 74 89 383494 66 12 57 51 86 383493 67 12 57 52 85 385905 67 1574 85 89 383492 68 12 60 56 85 385904 68 15 9 6 19 383491 69 12 38 41 81383490 70 12 51 49 84 383489 71 12 13 27 76 383488 72 12 24 38 82Control N/A N/A 52 51 86 Control N/A N/A 53 50 86

TABLE 9 ISIS 372648 Microwalk Compounds: Effect on Exon 7 Inclusion %Inclusion % Inclusion % Inclusion Target SMN2 Endogenous Endogenous ISIS# Site Length Minigene SMN2 SMN1 383470 92 18 8 12 63 383477 92 15 6 738 383469 93 18 29 26 89 383476 93 15 36 31 87 383487 93 12 11 16 79383468 94 18 31 26 88 383475 94 15 85 88 96 383486 94 12 44 41 91 38346795 18 14 13 82 383474 95 15 79 71 94 383485 95 12 63 60 93 372648 96 1570 57 93 383466 96 18 38 43 92 383484 96 12 65 56 92 383473 97 15 62 5394 383483 97 12 63 56 94 383472 98 15 41 46 93 383482 98 12 59 45 94383471 99 15 38 47 92 383481 99 12 42 46 92 383480 100 12 39 48 91383479 101 12 44 44 92 383478 102 12 47 41 93 Control N/A N/A 41 44 93Control N/A N/A 44 48 92

In accordance with previous results, treatment with ISIS 372642, ISIS372648 or ISIS 383475 led to a significant increase in exon 7 inclusion.In addition, ISIS 385905 was identified as a particularly effectivecompound for promoting exon 7 inclusion. To further evaluate ISIS 385905and ISIS 383475, dose-response and duration of action studies wereperformed. To determine the effect of oligonucleotide dose, HEK293 cellswere electroporated with either ISIS 385905 or ISIS 383475 at aconcentration of 0, 0.2, 0.5, 1, 2, 5, 10 or 20 μM. Sixty hours afterelectroporation, RNA was isolated and RT-PCR was performed as describedabove to determine the extent of exon 7 inclusion. The results,expressed as % inclusion of exon 7, are shown in Table 10.

TABLE 10 Dose-response of ISIS 385905 and ISIS 383475 0 0.2 0.5 1.0 2.05.0 10 20 ISIS # μm μm μm μm μm μm μm μm 385905 50 53 61 65 74 78 82 87383475 51 57 65 72 77 83 87 89

The results show a dose-dependent increase in exon 7 inclusion followingtreatment with either compound. To assess duration of action, HEK293cells were electroporated with 10 μM of either compound and RNA wasisolated and subjected to RT-PCR at day 0, 1, 2, 3, 4 and 5. Theresults, expressed as % inclusion of exon 7, are shown in Table 11.

TABLE 11 Duration of Action of ISIS 385905 and ISIS 383475 ISIS # Day 0Day 1 Day 2 Day 3 Day 4 Day 5 385905 49 80 83 82 85 79 383475 48 84 8988 89 83

These results demonstrate a significant increase in exon 7 inclusionfollowing treatment of either compound, and further show the compoundsare effective for at least five days.

Taken together, the results of the experiments detailed abovedemonstrate that antisense compounds having a target site (5′-mostnucleotide to which the compound binds) of nucleotides 64-68 or 94-97 ofSEQ ID NO: 1 are most effective at promoting exon 7 inclusion in SMN2transcripts. The target sites of these compounds overlap with predictedESS (exonic splicing silencer) elements, without having significantoverlap with predicted ESE (exonic splicing enhancer) elements. Thus,the antisense compounds described herein may function by blockingbinding of trans splicing factors to particular cis regulatory elements,thereby influencing splice site selection and specifically, inclusion orexclusion of exon 7 from SMN2 mRNAs.

Example 8 Effect of Compounds Targeting Exon 7 or Intron 7 on Inclusionof Exon 7

In previous examples herein, antisense compounds ISIS 372642, ISIS385905 and ISIS 383475, each of which target exon 7, were shown tosignificantly increase inclusion of SMN2 exon 7. These exon 7-targetedcompounds and a compound targeting SMN2 intron 7 (ISIS 387949) werecompared for their capacity to promote exon 7 inclusion. As described inprevious examples herein, HEK293 cells were electroporated with 10 μM ofoligonucleotide and RT-PCR was performed after two days to examinesplicing changes of endogenous SMN1 and SMN2. In comparison to controloligonucleotide, the compounds targeted to exon 7 exhibited asignificant increase in exon 7 inclusion, as expected. In addition, theintron 7 targeted compound led to incorporation of exon 7 in nearly allSMN1 and SMN2 mRNAs. These results suggest antisense compounds targetedto intronic sequences also contribute to incorporation of SMN2 exon 7.Intronic sequences also are known to contain splicing regulatoryelements (i.e. intronic splicing enhancers and intronic splicingsilencers), providing a possible mechanism of action for ISIS 387949.

Example 9 Systematic Mapping of Intronic Splicing Silencers (ISSs)

To further investigate whether antisense compounds targeting the intronsflanking exon 7 of SMN2 could alter inclusion of exon 7, such as byinterfering with intronic splicing silencers, compounds were designed totarget the 60 nucleotides of intron 6 (nucleotides 1-60 of SEQ ID NO: 1)or the 60 nucleotides of intron 7 (nucleotides 115-174 of SEQ ID NO: 1)immediately adjacent to exon 7. Antisense compounds targeting intron 6(ISIS 390636, ISIS 390637, ISIS 390638, ISIS 390639, ISIS 390640, ISIS390641, ISIS 390642, ISIS 390643, ISIS 390644 and ISIS 390645) or intron7 (ISIS 390646, ISIS 390647, ISIS 390648, ISIS 390649, ISIS 390650, ISIS390651, ISIS 390652, ISIS 390653, ISIS 390654 and ISIS 390655) are showabove in Table 1. Each compound was tested in three different assays toevaluate their effect on exon 7 inclusion: SMN2 minigene splicing incell-free extracts, SMN2 minigene splicing in transfected HEK293 cellsand splicing of endogenous SMN2 in HEK293 cells. The results obtainedfrom the three assays demonstrated that several antisense compounds wereable to increase inclusion of exon 7. In particular, ISIS 390644 andISIS 390648 were the most effect intron 6 and intron 7-targetedcompounds, respectively.

To further investigate the regions targeted by ISIS 390644 and ISIS390648, additional compounds were designed as microwalks around thesetarget sequences (see Table 1 for sequences). For these experiments,compounds 12 and 15 nucleotides in length were designed and tested inaccordance with the procedures detailed in previous examples herein.Compounds targeting the region of ISIS 390644 (intron 6) were tested inthe in vitro SMN2 minigene assay and endogenous SMN1/SMN2 assay inHEK293 cells. The results are shown in Table 12.

TABLE 12 Results of Microwalk of Intron 6 Compound ISIS 390644 %Inclusion % Inclusion Target SMN2 Endogenous ISIS # Site Length minigeneSMN2 393586 10 15 10 32 393587 9 15 18 44 393588 8 15 32 60 393589 7 1559 79 390644 6 15 65 75 393590 5 15 49 67 393591 4 15 20 46 393592 3 1522 44 393593 2 15 29 50 393594 13 12 20 44 393595 12 12 13 39 393596 1112 15 44 393597 10 12 13 39 393598 9 12 17 48 393599 8 12 30 64 393600 712 28 62 393601 6 12 44 63 393602 5 12 29 46 Control N/A N/A 22 43

As shown in Table 12, antisense compounds having a target site ofnucleotides 5-8 (SEQ ID NO: 1) results in the greatest percentage oftranscripts containing exon 7. These findings suggest this region ofintron 6 contains an intronic splicing silencer, which normallyfunctions to inhibit inclusion of exon 7. Upon blockade of thisregulatory element, splice site selection is altered to promote exon 7inclusion.

Compounds targeting the region of 390648 (intron 7) were assayed usingthe SMN2 minigene in vitro and in transfected HEK293 cells and tested inthe endogenous SMN2 splicing assay in HEK293 cells. The results areshown in Table 13.

TABLE 13 Results of Microwalk of Intron 7 Compound ISIS 390648 %Inclusion % Inclusion % Inclusion Target SMN2 in vitro SMN2 EndogenousISIS # Site Length minigene minigene SMN2 393603 129 15 43 51 76 393604128 15 46 75 97 393605 127 15 57 97 100 393606 126 15 56 97 100 390648125 15 53 98 100 393607 124 15 67 100 100 393608 123 15 75 100 100393609 122 15 60 97 100 393610 121 15 54 58 78 393611 132 12 41 17 40393612 131 12 39 30 58 393613 130 12 42 43 64 393614 129 12 48 43 64393615 128 12 38 36 44 393616 127 12 38 30 90 393617 126 12 36 30 92393618 125 12 44 71 97 393619 124 12 69 92 97 Control N/A N/A 28 23 44

While all compounds led to an increase in exon 7 inclusion, compoundswith target sites between nucleotides 121 and 129 (SEQ ID NO: 1) weremost effective.

Select compounds targeting intron 7 were further evaluated for SMN2 exon7 inclusion following transfection at a low oligonucleotide dose of 0.1μM. As previously described herein, HEK293 cells were electroporatedwith ISIS 393605, ISIS 393606, ISIS 390648, ISIS 393607, ISIS 393608,ISIS 393609, ISIS 393617, ISIS 393618 or ISIS 393619 and levels ofendogenous SMN2 splice products were determined. The results are shownin Table 14.

TABLE 14 Effect on SMN2 Exon 7 Incorporation Following Low-DoseTreatment % Inclusion Target Endogenous ISIS # Site Length SMN2 393605127 15 56 393606 126 15 58 390648 125 15 60 393607 124 15 60 393608 12315 63 393609 122 15 53 393617 126 12 51 393618 125 12 51 393619 124 1257 Control N/A N/A 49

As shown in Table 14, even at a very low dose, antisense compoundstargeting intron 7 are effective at promoting inclusion of exon 7. Takentogether, these results suggest the region near the 5′ end of intron 7(encompassing nucleotides 121-129 of SEQ ID NO: 1) contains an intronicsplicing silencer.

1. An antisense oligonucleotide targeted to intron 6, exon 7 or intron 7of a nucleic acid molecule encoding SMN2, wherein said oligonucleotideis 12 to 20 nucleotides in length and comprises a 2′-O-methoxyethylsugar modification at each position.
 2. The oligonucleotide of claim 1which is 12 nucleotides in length.
 3. The oligonucleotide of claim 1which is 15 nucleotides in length.
 4. The oligonucleotide of claim 1which is 18 nucleotides in length.
 5. The oligonucleotide of claim 1which is targeted to an intronic splicing silencer element.
 6. Theoligonucleotide of claim 1 which is targeted to an exonic splicingsilencer element.
 7. The oligonucleotide of claim 1 which is targeted tointron
 6. 8. The oligonucleotide of claim 7, wherein the target site ofsaid oligonucleotide is nucleotide 5, 6, 7 or 8 of SEQ ID NO:
 1. 9. Theoligonucleotide of claim 7, wherein the nucleotide sequence of saidoligonucleotide comprises at least an 8-nucleobase portion of SEQ ID NO:6, 7, 8, 9, 10, 11, 12 or
 13. 10. The oligonucleotide of claim 1 whichis targeted to exon
 7. 11. The oligonucleotide of claim 10 wherein thetarget site of said oligonucleotide is nucleotide 64, 65, 66, 67, 68,94, 95, 96 or 97 of SEQ ID NO:
 1. 12. The oligonucleotide of claim 10,wherein the nucleotide sequence of said oligonucleotide comprises atleast an 8-nucleobase portion of SEQ ID NO: 37, 38, 41, 59, 62, 63, 64,66, 67 or
 68. 13. The oligonucleotide of claim 1 which is targeted tointron
 7. 14. The oligonucleotide of claim 13 wherein the target site ofsaid oligonucleotide is nucleotide 121, 122, 123, 124, 125, 126, 127,128 or 129 of SEQ ID NO:
 1. 15. The oligonucleotide of claim 13, whereinthe nucleotide sequence of said oligonucleotide comprises at least an8-nucleobase portion of SEQ ID NO: 79, 80, 81, 82, 83, 84, 85, 86, 87,89, 91 or
 93. 16. A method of promoting inclusion of exon 7 in SMN2transcripts in a cell, tissue or organ, comprising contacting said cell,tissue or organ with the antisense oligonucleotide of claim
 1. 17. Themethod of claim 16 wherein said oligonucleotide is targeted to anintronic splicing silencer element.
 18. The method of claim 16 whereinsaid oligonucleotide is targeted to an exonic splicing silencer element.19.-22. (canceled)
 23. A pharmaceutical composition comprising theantisense oligonucleotide of claim 1.